A chemical signal in human female tears lowers aggression in males

Rodent tears contain social chemosignals with diverse effects, including blocking male aggression. Human tears also contain a chemosignal that lowers male testosterone, but its behavioral significance was unclear. Because reduced testosterone is associated with reduced aggression, we tested the hypothesis that human tears act like rodent tears to block male aggression. Using a standard behavioral paradigm, we found that sniffing emotional tears with no odor percept reduced human male aggression by 43.7%. To probe the peripheral brain substrates of this effect, we applied tears to 62 human olfactory receptors in vitro. We identified 4 receptors that responded in a dose-dependent manner to this stimulus. Finally, to probe the central brain substrates of this effect, we repeated the experiment concurrent with functional brain imaging. We found that sniffing tears increased functional connectivity between the neural substrates of olfaction and aggression, reducing overall levels of neural activity in the latter. Taken together, our results imply that like in rodents, a human tear–bound chemosignal lowers male aggression, a mechanism that likely relies on the structural and functional overlap in the brain substrates of olfaction and aggression. We suggest that tears are a mammalian-wide mechanism that provides a chemical blanket protecting against aggression.

Major comments 1. * The introduction focuses on biological mechanisms of chemical signaling (e.g., the role of ESP1), which is interesting and somewhat relevant.However, given the nature of the study, shifting the introduction's focus toward behavioral outcomes, especially in humans, would provide helpful context.For instance, the authors could briefly report behavioral and fMRI findings from Gelstein et al., 2011 (from the same group), Mishor et al., 2021 (also from the same group), and Oh et al., 2012, and discuss the known fMRI correlates of aggression.
We have modified the introduction to now also discuss the human behavioral and brain response to tears, and to manipulations of aggression.Moreover, we discuss Mishor et al and related materials in the discussion.
The authors have adequately addressed my comment.
2. * Behavioral findings from experiment 3 should be reported in a similar manner to those from experiment 1 (i.e., by comparing APR values across conditions for the entire group, and plotting the data in figures similar to those in Figure 1 We concur, and have changed the relevant figures accordingly.I appreciate the authors' transparency in addressing this comment in the manuscript text, but can they move the supplementary figure (or at least panels A-B) into the main manuscript?These could appear as new panels in Figure 1 (alongside behavioral results from Experiment 1), or in Figure 3 (alongside MRI results from Experiment 3).Also, if the authors think it's important to include their theory about order affecting behavior in the scanner, they should briefly mention it in the main text (even in the figures are relegated to the supplement).These changes could serve to curb unrealistic expectations regarding the size of this effect and/or emphasize the need to consider fMRI and ordering components for future work.Finally, can the authors adjust the statistical analysis section to account for the one-tailed nature of their new t-test?: "Normally distributed data were analyzed using ANOVAs followed by planned two-tailed t-tests, or onetailed t-tests when explicitly noted." 3. Although it's plausible that order effects could be accentuated in the MRI scanning context, properly counterbalancing the conditions should account for this issue.However, based on data file 4, it seems that the groups were no longer well-balanced following exclusions, with 11 participants in the saline-first group, and 15 participants in the tears-first group.If order plays a significant role in modulating aggression as the authors suggest, this is a significant problem, since differences in fMRI correlates of aggression could be driven by the order of conditions, rather than the chemical stimulus applied.Ideally, the authors would recruit more participants to balance the groups, or at least account for the imbalance somehow in their models.This is a keen observation with which we concur.To address this potential problem and balance the groups, we conducted the following analyses: We have 9 fMRI participants who had saline-first.Thus, a balanced group from this perspective is reduced to 18 participants, which is borderline in power.To overcome this, we conducted two analyses.First, using a bootstrap approach, we randomly selected balanced groups of 18 participants 10,000 times, and each time conducted the analysis to create a distribution.Had we done this on the entire brain it would have taken a few months to run, therefore we limited this analysis to the two ROIs we reported in the study, namely the AIC and PFC.In a second analysis we made a one-time selection of a balanced group of 18, and conducted full brain analysis.Here we had to first decide which tears-first participants we ignore, and did this by removing the low AGQ scoring individuals to maintain two AGQ balanced groups.Both of these tests confirmed our previous findings.The bootstrap analysis revealed an average t(17) = 2.414, p-value = 0.037 for the left AIC, and an average t(17) = 3.815, p-value = 0.0015 for the PFC.New Supplementary Figure 6A-B provides additional details.The whole brain analysis of the counterbalanced group (n=18) is available in new Supplementary Figure 6C.Furthermore, the correlation between behavior and brain response in the ROIs remained consistent even in the smaller group, as shown in Supplementary Figure 6D-E.I appreciate the authors' thorough approach in addressing my comment.In the main manuscript, the authors introduce these control analyses before bringing up that AIC and PFC emerged as significant in the initial analysis, which might be confusing for readers.I'd recommend revising the order of information for clarity.Also, could the authors implement the same color bar scale for the supplementary figure as the main text figure for ease of comparison?Otherwise, the authors have adequately addressed my comment.
4. * Although results from this study could be related to COVID-19, this is very speculative.It's not clear that anosmia in COVID patients results in a parallel deficit in "odorless" chemical signaling, and there are a host of other (probably more salient) reasons why aggression might manifest in individuals with a complex illness.Drawing conclusions about men in particular seems inappropriate, given that there's no gender comparison in this study.Perhaps the authors could bring up the COVID-19 link, but in a more measured way, and without emphasizing it.

Agreed -removed completely
The authors have adequately addressed my comment.
Minor comments 5. * Were participants asked whether they believed their PSAP opponent was human?If not, this should not be assumed in the results section.
To ensure the integrity of the experiment's paradigm, we did not ask participants directly if they believed they were playing against a real opponent before finishing collecting the data.Instead, we collected information indirectly by asking participants about their thoughts and feelings towards their opponent, both verbally and through a questionnaire at the end of the experiment.We only excluded one participant who expressed doubt about his opponent's human identity, as social interaction is a crucial element of our study on social chemosignals.We now added further clarification in the methods section (see Questionnaires).This makes sense.Can the authors modify the description of the paradigm to reflect this?: "In brief, in the PSAP participants play a monetary game with an opponent they are told is human, but is in fact a computer algorithm."6. * It's stated that participants did not know they were sniffing tears during the sniff-jar procedure.What were the participants told, if anything, about the stimulus?
Our IRB document that participants sign, and are supposed to read before participation (although few if any read it carefully) noted that "as a participant you may receive assorted odors, including body odors, and at various intensities, above and below threshold".In other words, they generally know (and agree) that sniffing body odors is a "possibility" they may encounter in the study, but this is not up front in their attention.
The authors have adequately addressed my comment.
7. * Could the authors include bar plots to illustrate the mean pleasantness, intensity, and familiarity for tears vs. saline in Figure 1? There's also an inconsistency on the plot (which references experiments 1 and 2) and the legend and supplementary data file (which reference experiments 2 and 3) -from reading the manuscript, I believe that all of these should reference experiments 1 and 3. Most importantly though, the idea that the tears are "odorless" is not supported by the data, since participants rate the tear stimuli as moderately intense.It would be more accurate to say that previous studies (e.g., Gelstein et al., 2011) have demonstrated that tear and saline stimuli cannot be discriminated based on odor, which implies that the chemical signal contained in tears is odorless.The data collected here support this idea (since there aren't meaningful differences in ratings across stimuli), but it is not tested directly.
Thanks for drawing our attention, we fixed this typo and added a bar plot in Supplementary Figure 2A-C.We would like to stress that the tears are perceptually odorless.The "moderate intensity" ratings applied by participants are merely because human raters rate a non-stimulus as moderate in such tests.This is clearly seen in that saline was also rated in the same way, and saline is clearly odorless.It has no VOC in it.We could have presented the data as "difference from saline", and then tear intensity would be at zero, yet we opt to show the rating data as is.Indeed, both scatter and bar plots show that tear ratings did not differ from those of trickled saline in all tested parameters, including intensity.Moreover, both stimuli (trickled saline and tears) odor descriptor ratings did not differ from blank (not trickled) saline solution (we added this information to the text and to Supplementary Figure 2D-F).
I do not think the authors have corrected the typo in the legend of Figure 1, which reads: "Each dot is the average of 10 sniffs by a given participant, light-colored dots are from Experiment 2 (n=22), and dark dots are from Experiment 3 (n=24)."Also, I'm confused as to why the odor ratings are so much higher across the board for Experiment 3 compared to Experiment 1 -is this due to the normalization approach?Finally, I take the authors' points regarding the perceptually odorless nature of tears.It's interesting that humans tend to rate non-stimuli as moderate.
8. * The APR is a ratio, but several participants have a score of 0 for one or both conditions.What does a 0 value mean in this case?And do the numbers following the mean APR (e.g., 1.67+/-1.7 APR) represent the standard error?
A score of zero means no aggressive response (0 revenge response divided by the number of provocations).The numbers following the mean APR are standard deviation (SD) and not standard error, we added this clarification to the text.
The authors have adequately addressed my comment.9. * It does not seem appropriate to include OR1A1 in the olfactory receptor analysis, given that it does not meet the established criteria, and there are other ORs that are similarly close to the cutoff, but are not included (e.g., OR5K1).If the authors feel that it's important to include this receptor, then they should explain why in the main text (beyond stating general interest), and this should be framed as a separate exploratory analysis (after the main analysis).Could the authors also provide justification for moving forward with receptors that are activated, but not inhibited, by the stimuli (given that inhibition could also occur in a dose-dependent manner)?
We concur that retaining OR1A1 in the manuscript does not meet strict statistical inclusion criteria, and therefore we removed it.It was indeed there because we have an extended interest in it from a different study, but this is indeed irrelevant.Finally, two ORs, 2W1 and 10A4, were